The separation of the 34S isotope was carried out by isotopic exchange between sulfurous acid in solution and bisulfite anions adsorbed on a quaternary ammonium (Dowex 1X8 and Dowex 2X8, 100-200 mesh) packed in six anion exchange columns. Each resin column packed with the anion exchange resin had a height of 115 cm and 2.2 cm in diameter. The columns were connected in series during the displacement of bisulfite bands. For the experiments, a band of bisulfite was fixed to the anion resin, initially in the hydroxyl ion form, and subsequently eluted with 0.2, 0.3, 0.4, or 0.6 mol L-1 HCl solution. The hydrochloric acid solution was kept under nitrogen at 245 kPa, in order to prevent the evolution of gases and the oxidation of the bisulfite band. The experiments with the Dowex 1X8 resin showed that the best results in terms of maximum enrichment (18.66 atom %34S) and total34S accumulated mass (161.7 mg, excess) were obtained by elution of the bisulfite band for 50 m with 0.2 and 0.3 mol L-1 HCl, respectively. The depleted portion of the band was periodically replaced with natural bisulfite, at 10 m intervals during elution.
isotopic enrichment; stable isotope; enriched sulphur; 34S
A separação do isótopo 34S foi obtida pela reação de troca isotópica entre solução de ácido sulfuroso e ânions bissulfito adsorvidos em resinas aniônicas tipo amônio quaternário (Dowex 1X8 e Dowex 2X8, malha 100-200) acondicionadas em seis colunas de resina de troca aniônica. Cada coluna de resina possuia 130 cm de comprimento com diâmetro de 2,2 cm. As colunas foram conectadas em série durante o deslocamento da banda de bissulfito. Para a experimentação, a banda de bissulfito fixada na resina aniônica, inicialmente na forma OH-, foi eluída subsequentemente com 0,2, 0,3, 0,4 ou 0,6 mol L-1 HCl em solução. Ácido clorídrico em solução foi introduzido juntamente com nitrogênio sob pressão de 245 kPa, para impedir durante o deslocamento, a liberação de gases e a oxidação da banda de bissulfito. Os experimentos utilizando a resina Dowex 1X8 mostraram que os melhores resultados em termos de máximo enriquecimento (18,66 % em átomos de34S) e da massa total acumulada de34S (161,7 mg, excesso) foram obtidos eluindo a banda de bisssulfito por 50 m com HCl 0,2 e 0,3 mol L-1, respectivamente. A substituição da parte empobrecida da banda com bissulfito natural foi efetuada a cada deslocamento de 10 m.
Stable sulfur isotope fractionation by anion exchange chromatography. production of compounds enriched in 34S
José Albertino BendassolliI; Paulo Cesar O. TrivelinI; Francisco Carneiro JuniorII
ICentro de Energia Nuclear na Agricultura - CENA, Universidade de São Paulo - USP, Av. Centenário, 303, C.P. 96, 13.400-970 Piracicaba - SP, Brazil
IIUniversidade Metodista de Piracicaba - UNIMEP, Rua Rangel Pestana, 762, C.P. 68 13.400-901 Piracicaba - SP, Brazil
The separation of the 34S isotope was carried out by isotopic exchange between sulfurous acid in solution and bisulfite anions adsorbed on a quaternary ammonium (Dowex 1X8 and Dowex 2X8, 100-200 mesh) packed in six anion exchange columns. Each resin column packed with the anion exchange resin had a height of 115 cm and 2.2 cm in diameter. The columns were connected in series during the displacement of bisulfite bands. For the experiments, a band of bisulfite was fixed to the anion resin, initially in the hydroxyl ion form, and subsequently eluted with 0.2, 0.3, 0.4, or 0.6 mol L-1 HCl solution. The hydrochloric acid solution was kept under nitrogen at 245 kPa, in order to prevent the evolution of gases and the oxidation of the bisulfite band. The experiments with the Dowex 1X8 resin showed that the best results in terms of maximum enrichment (18.66 atom % 34S) and total 34S accumulated mass (161.7 mg, excess) were obtained by elution of the bisulfite band for 50 m with 0.2 and 0.3 mol L-1 HCl, respectively. The depleted portion of the band was periodically replaced with natural bisulfite, at 10 m intervals during elution.
Keywords: isotopic enrichment, stable isotope, enriched sulphur, 34S
A separação do isótopo 34S foi obtida pela reação de troca isotópica entre solução de ácido sulfuroso e ânions bissulfito adsorvidos em resinas aniônicas tipo amônio quaternário (Dowex 1X8 e Dowex 2X8, malha 100-200) acondicionadas em seis colunas de resina de troca aniônica. Cada coluna de resina possuia 130 cm de comprimento com diâmetro de 2,2 cm. As colunas foram conectadas em série durante o deslocamento da banda de bissulfito. Para a experimentação, a banda de bissulfito fixada na resina aniônica, inicialmente na forma OH-, foi eluída subsequentemente com 0,2, 0,3, 0,4 ou 0,6 mol L-1 HCl em solução. Ácido clorídrico em solução foi introduzido juntamente com nitrogênio sob pressão de 245 kPa, para impedir durante o deslocamento, a liberação de gases e a oxidação da banda de bissulfito. Os experimentos utilizando a resina Dowex 1X8 mostraram que os melhores resultados em termos de máximo enriquecimento (18,66 % em átomos de 34S) e da massa total acumulada de 34S (161,7 mg, excesso) foram obtidos eluindo a banda de bisssulfito por 50 m com HCl 0,2 e 0,3 mol L-1, respectivamente. A substituição da parte empobrecida da banda com bissulfito natural foi efetuada a cada deslocamento de 10 m.
Sulfur is one of the essential elements for plant and animal life. In plants it has many functions besides being part of amino acids and proteins: in the form of SO4-2it activates enzymes, as SH it is an active group in enzymes and coenzymes (lipoic acid, thiamine, biotin), in the process of photosynthesis it participates in chlorophyll synthesis, CO2 absorption, carboxylase activity, ribulose-2P, and the phosphorylation reaction, and it is essential in the N fixation process in legumes1.
More studies are needed to more precisely define the importance of sulfur in plant nutrition, since little attention has been given to this matter up to now. Although reports about sulfur deficiency in cultivated plants around the world, and especially in Brazil are common, few studies on this nutrient have been carried out . There is an urgent need for research in this field2.
The isotopic technique has proven to be very valuable for elucidating aspects related to sulfur as a plant nutrient, and also for evaluating its utilization by cultivated plants. The radioisotope 35S has been used as a tracer in studies of the soil-plant system, due to its availability, relatively low cost, and non-sophisticated analytical technique. 35S has a decay of b- 0.167 Mev and a half-life of 88 days3. Currently, there is a tendency, at the international level, to encourage the use of non-radioactive techniques in isotopic tracer research, especially in field experiments.
The use of 34S as a sulfur tracer allows for the development of experiments without time restraints. Because the experimental material is not exposed to radiation, special safety precautions are not needed. Hence, the use of the tracer technique with the 34S stable isotope is an alternative for evaluations of sulfur dynamics in the soil-plant system. Recently, laboratories in the USA have produced compounds enriched with the 34S stable isotope. Because of this and also due to progress in the analytical technique using mass spectrometry, the first studies on the 34S stable isotope using marked compounds have been developed4,5.
Although the studies using compounds enriched with 34S are recent, the results obtained show tremendous potential and are a good alternative in studies using this important nutrient for plants, especially in long term projects.
It is fundamental when using the 34S tracer in research projects that the method of isotopic separation and the production of labeled compounds in the isotope be dominated. It is feasible to supply Brazilian researchers in soil, environmental and biological sciences in general with this product at a reasonable price, one which is lower than the foreign product, and similar to that of the 35S radioisotope.
The goal of this work is to study the method of 34S enrichment by anion exchange chromatography in resin columns. There is little information in the literature on the technical and operational details of the resin column system for the enrichment of 34S, nor much related to the simultaneous separation of the other stable sulfur isotopes and isotopic analysis. Hence, different experimental conditions have been evaluated (e.g. resin type, eluent, chemical concentration, and band displacement) in order to obtain 34S enrichments up to of 20 atom %. Studies have also been carried out to obtain simultaneous separation of the other less abundant stable sulfur isotopes (33S and 36S). After obtaining 34SO2(aq) in the isotopic enrichment columns, it is possible to prepare other inorganic compounds enriched with the isotope (NaH34SO3, Na234SO3, Na234SO4,(NH4)234SO4).
Anionic resins of the strong base divinylbenzene polystyrene type (Dowex 1X8 and 2X8, 100-200 mesh) with the following characteristics were used: polymerization with 8% divinylbenzene (% DVB); functional group CH2N+(CH3)3 for Dowex 1X8 and CH2N+(CH3)2 (C2H4OH) for Dowex 2X8; anionic exchange capacity in Cl- of 3.2 mmol g-1(dry resin) and 1.4 mmol g-1 (moist resin); resin diameter (100-200 mesh): 74-149 mm (U.S. standard).
Column system and apparatus
The enrichment system consisted of six columns of acrylic, 130 cm in length and having a 2.2 cm internal diameter, filled with 100-200 mesh anionic resin (Dowex 1X8 or Dowex 2X8), up to a height of 115 cm, in the form of Cl-. Each column had in lower section an acrylic cylindrical apparatus connected to the tube by a screw. On the base of this acrylic part two stainless steel screens, 50- and 300-mesh, and one 300-mesh propylene screen were attached in sequence. Above them, a neoprene O-ring, made of C 3002-7B, was placed to ensure sealing. In superior section of the column, where the chemical solutions were poured, a PVC piston with two neoprene rings was adapted. When moved upwards or downwards this PVC piston maintained the level of the solution in the column constant, at a few mm above the surface of the resin bed. The columns were connected in series with the aid of 1 mm internal-diameter polyvinyl chloride tubing.
The containers with sodium hydroxide, sodium bisulfite, and deionized water were placed 4 m above the columns, and the container with hydrochloric acid eluent solution was leveled with the columns. The flux of hydrochloric acid through the resin layers in the columns was effected with the aid of a system working under nitrogen pressure, at approximately 245 kPa. The entire system can be observed in Fig. 1.
For the isotopic analysis, a mass spectrometer model ATLAS-MAT CH-4 was used, having the following features: analyzer tube curvature radius of 120°; ionization by electronic impact; admission system with molecular flux; simple ion collector, Faraday-type cup; and scanning analysis system.
During the development of this study the isotopic separation of 34S was investigated using two types of resins and four concentrations of hydrochloric acid (0.2, 0.3, 0.4, and 0.6 mol L-1) in five experiments (I to V), as shown in Table 1.
At the beginning of the experiments the resins were regenerated to the OH- form with a solution of 2 mol L-1 sodium hydroxide, at a flow rate of 3.8 cm3 min-1, followed by rinsing the resin layers in each column with deionized water, and fixing a band of sodium bisulfite solution in one of the columns (4 L of solution 0.15 mol L-1 column-1). This band was then eluted with an HCl solution, at a rate of 3.8 cm3 min-1, having a second column connected in series. While the band displacement occurred, it was confined between two well-defined limits. These limits were made at front of the band by the equilibrium constants of the ionic exchange reactions between HSO3- in solution and adsorbed hydroxyl anions, and at the rear of the band by the reaction between Cl- in solution and adsorbed HSO3-.
During displacement (elution), samples were collected along the band in order to evaluate the 34S concentrations. The sampling procedure consisted of collecting small fractions of sulfurous acid at different positions along the band length, in one displacement column. The samples taken had equivalent amounts or twice as much sodium hydroxide as desired, making it possible to obtain sodium bisulfite or sodium sulfite. For the isotopic analysis by mass spectrometry, the samples were prepared by transforming the sodium bisulfite or sodium sulfite into sulfur dioxide under vacuum6. The region of the band with a normal isotopic concentration of 34S (natural concentration threshold) was maintained at a minimum of 20% of the band. This control was done by the addition, when necessary, of a solution of 0.15 mol L-1 sodium bisulfite with 34S natural concentration, following the discard of the same amount of band solution depleted of the isotope (with a concentration of less than 4.2 atom % 34S).
In this process, the equilibrium between the sulfurous acid solution and HSO3- ions adsorbed to the anion exchange resin (HSO3- - H2SO3 system), for the fractionation of 33S and 32S, 34S and 32S, or 36S and 32S by ionic exchange in resin columns, is represented by Eq. 17.
where *S represents the isotopes 33S , 34S, or 36S, and (r) and (s) stand for resin and solution, respectively.
Results and Discussion
A summary of the main results is given in Table 2, showing that Experiments II and III had the best results. The data shown in Table 2 indicate that the best results for maximum enrichment (18.66 atm% 34S) and total 34S accumulated mass (161.7 mg, excess) were obtained by 50 m of elution of the bisulfite band with 0.2 and 0.3 mol L-1 acid, respectively. The experimental data proved that in experiments II and III the enriched band was established in the last 20 and 35 cm, respectively. These results, which can be observed in Fig. 2 and Table 2, show that a higher accumulation of 34S mass occurs in Experiment III (161.7 mg), when compared to Experiment II. One of the most relevant parameters is the accumulation rate (excess) of the isotope 34S (mg h-1). The data in Table 2 indicate that this rate is approximately 20% higher in Experiment III (HCl 0.3 mol L-1) when compared to Experiment II (HCl 0.2 mol L-1).
Under the conditions used for experiments II and III, the enrichment curves for 34S (Fig. 2) were obtained after 50 m of bisulfite band elution. It is possible to obtain from Experiments II and III, 6.46 and 7.37 mmol sulfurous acid respectively as a column effluent, which corresponds to 1 linear centimeter of the band (at the rear of the band). Table 2 also shows that Experiment V had the least favorable results in terms of 34S isotope enrichment.
Analyzing Table 2, it can also be observed that Experiment I presented results comparable to Experiments II and III. However, the use of the resin Dowex 2X8 in this experiment was inadequate under these experimental conditions, especially due to difficulties in visualizing the band limits, causing isotopic mixtures. This made it impossible to proceed with the experiment with the distance of the band elution superior to 30 m. In Experiments IV and V the distance of the band elution was achieved only up to 10 m (Table 2), due to the concentrations of hydrochloric and sulfurous acid, which constantly caused corrosion of the stainless steel at the column bases. This caused many difficulties for the enrichment process including resin loss, discontinuance of the displacement band, isotopic mixtures, and chance of enrichment band loss, among others.
In Experiment II, the process had continuity (routine) after 50 m of band elution. It was possible to obtain for every 14 m from the last centimeter of band, an average of 6.79 mmol of sulfurous acid enriched at 16.76 atoms % in 34S. This amount of sulfurous acid can be converted to 706, 855, or 964 mg of sodium bisulfite, sodium sulfite, or sodium sulfate, respectively. Sodium sulfate can be obtained by the oxidation of the solution of sodium sulfite with oxygen.
After 50 meters of band elution the sulfurous acid solution was sampled at about 12 cm from the rear limit. This had the objective of evaluating the presence of sulfate which could originate from sulfite oxidation. The concentrations of sulfate and sulfite in solution were 0.43 and 35.88 mg cm-3, respectively. These results indicate low oxidation, since about 1.2% of the initial sulfite was oxidized. There is also an indication that the system under nitrogen pressure in the reservoir of the HCl eluent solution was efficient in terms of preventing the dissolved air in solution (especially O2) from favoring the oxidation reaction.
In Experiment II the isotopic concentration of the sulfur isotopes was also determined. The ratio of 33S, 34S and 36S isotope concentrations at 1 centimeter from the back of the band limit to the natural abundance values were 2.0, 4.0, and 5.3, respectively. These values allow for the inference that the fractionation values (a) for the isotopes 33S, 34S, and 36S in equilibrium with the most abundant sulfur isotope (32S) should obey the following order: α36S > α 34S > α33S.
Based on the results obtained it can be concluded that: a) the use of Dowex 2X8 resin for 34S isotopic enrichment is not adequate under the experimental conditions used, due to the problem of visualization of the rear of the band; b) it is possible to obtain sulfurous acid with 34S enrichment of 20 atom % in anionic resin columns of Dowex 1X8, by displacing the bisulfite band with HCl 0.2 or 0,3 mol L-1 eluent solution; c) by using the proposed system of resin columns, after 50 m of elution, 6.46 mmol (17.33 atom % of 34S) and 7.37 mmol (16.93 atom % of 34S) of sulfurous acid are obtained in the last linear centimeter of the band with the use of 0.2 and 0.3 mol L-1 HCl, respectively. The average rate of excess accumulation of 34S is 0.16 and 0.19 mg per hour of elution with 0.2 and 0.3 mol L-1 acid, respectively; d) in routine, after the first 50 m of band elution, under experimental conditions, it is possible to obtain 6.79 mmol of sulfurous acid for every 14 m of band elution with approximately 17 atom % in 34S, in the last linear centimeter at the rear of the band; e) the resin column system, working at a pressure of 245 kPa under nitrogen atmosphere was able to control the release of gases and especially sulfite oxidation.
The authors thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for the valuable financial support for this research (Process: 90/4365-0).
Received: October 31, 1995; September 7, 1996
FAPESP helped in meeting the publication costs of this article
- 1. Malavolta, E. Nutrição Mineral; Ferri, M.G. (coord) In Fisiologia Vegetal 1, EPU, São Paulo, 1979; p 97.
- 2. Bissani, C.A.; Tedesco, M.J. O Enxofre no Solo In: Reunião Brasileira De Fertilidade Do Solo, 17, Londrina, l986. Bordert C.M; Lantmann, A.F.; Ed. Londrina, Embrapa/CNPSO, IAPAR, SBCS, 1988; p 11.
- 3. Weast, R.C. Handbook of Chemistry and Physics 50 ed., Cleveland, Chemical Rubber, 1969; p B-273.
- 4. Hamilton, S.D.; Chalk, P.M.; Unkovich, M.J.; Smith, C.J. Applied Radiation and Isotopes 1991, 42 (11), 1099.
- 5. Awonaike, K.O.; Danso, S.K.A.; Zapata, F. Plant and Soil 1993, 155/156, 325.
- 6. Bendassolli, J.A. Separação dos isótopos de enxofre em colunas de resina de troca aniônica. Enriquecimento isotópico de 34S, Doctor Thesis, Centro de Energia Nuclear na Agricultura (CENA/USP), Piracicaba, SP, Brasil; l994.
- 7. Forberg, S.; Barnevik, W.; Fogelstrom-Fineman, I.; Westermark, T.; Ubisch, H.V. Enrichment of heavy sulphur isotopes with an anion exchanger. In: International Symposium on Isotope Separation. l957. Proceedings, edited by J. Kistemaker; J. Bigeleisen; A.O.C. Nier. Amsterdam, North-Holland Publishing, 1958; p 243.
Publication in this collection
04 Oct 2011
Date of issue
31 Oct 1995